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Article Open Access

Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma

  • Authors:
    • Sonexai Kidoikhammouan
    • Panupong Mahalapbutr
    • Prasertsri Ma‑In
    • Worachart Lert‑Itthiporn
    • Raksawan Deenonpoe
    • Utid Suriya
    • Sopit Wongkham
    • Wunchana Seubwai
  • View Affiliations / Copyright

    Affiliations: Biomedical Sciences Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, Center for Translational Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
    Copyright: © Kidoikhammouan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 52
    |
    Published online on: March 3, 2026
       https://doi.org/10.3892/br.2026.2125
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Abstract

Gemcitabine resistance remains a significant problem in cholangiocarcinoma (CCA) treatment. The present study aimed to identify novel molecular targets associated with gemcitabine resistance in CCA using integrated bioinformatics, molecular docking, and experimental validation. Commonly differentially expressed genes (DEGs) were identified based on three public transcriptomic datasets (GSE116118, GSE208659, and GSE140077) from stable gemcitabine‑resistant cancer cell lines. Expression levels of selected upregulated genes were validated in Thai patients with CCA using the GEO datasets. Further validation was performed using real‑time PCR on parental and gemcitabine‑resistant CCA cell lines (KKU‑213A/KKU‑100 and KKU‑213A‑GemR/KKU‑100‑GemR). The 3D structure of the candidate protein was modeled using AlphaFold3 and refined by molecular dynamics simulations. Druggable pockets were predicted using CavityPlus. Virtual screening of 5,396 drug‑repurposing compounds was performed using AutoDock Vina to identify potential inhibitors of the candidate protein. In total, 14 DEGs were commonly upregulated in three datasets from stable gemcitabine‑resistant cancer cell lines. A total of six genes, including RAB1B, JAG1, PACS1, ANKS6, SF3B4, and RTKN2, were significantly elevated in CCA tissues compared with adjacent normal tissues. Reverse transcription‑quatitative PCR confirmed that RTKN2 was significantly overexpressed in two stable gemcitabine‑resistant CCA cell lines, KKU‑213A‑GemR and KKU‑100‑GemR. Pathway analysis of RTKN2‑associated genes revealed enrichment in mitochondrial and metabolic processes, including suppression of the TP53‑regulated metabolic pathway. Molecular docking analysis identified several candidate compounds with strong binding affinities to RTKN2 (‑10.49 to ‑11.47 kcal/mol), including NKP608, tegatrabetan, umbralisib, vepdegestrant, and MK‑3207. RTKN2 was identified as a novel and druggable molecular target potentially contributing to gemcitabine resistance in CCA. Targeting RTKN2 may offer a promising approach to overcome chemoresistance and improve outcomes for patients with CCA.
View Figures

Figure 1

Identification of DEGs in
gemcitabine-resistant cancer cell lines. (A) Volcano plots of DEGs
from three stable gemcitabine-resistant cancer cell lines. The
datasets included GSE116118, GSE208659, and GSE140077. The x-axis
represents the log2 fold change, and the y-axis
represents the negative logarithm (base 10) of the adjusted
P-value. Red dots represent significantly upregulated genes, blue
dots represent significantly downregulated genes, and black dots
represent genes without significant changes. (B) Venn diagram
displaying the overlap of DEGs in three transcriptomic datasets.
Numbers within the sections indicate the count of DEGs. The green
color represents the DEGs from the cholangiocarcinoma cell line
(GSE116118), blue represents the pancreatic cancer cell line
(GSE140077), and pink represents the gallbladder cancer cell line
(GSE208659), respectively. The central number of all circles
overlapped demonstrates the common DEGs between the datasets. DEGs,
differentially expressed genes.

Figure 2

Validation of common upregulated
genes associated with gemcitabine resistance in CCA tissues and
cell lines. (A) Box plots show the mRNA expression levels of RAB1B,
JAG1, PACS1, ANKS6, SF3B4, and RTKN2 in CCA and normal tissues. The
Mann-Whitney U test was used to evaluate the expression differences
between CCA and normal tissue. (B) PCR analysis of six common
upregulated genes associated with gemcitabine resistance in stable
gemcitabine-resistant CCA cell lines. The data are expressed as the
mean ± SD from three independent experiments. (C) RTKN2 mRNA
expression after gemcitabine treatment in CCA cell lines. KKU-213A
and KKU-100 cells were treated with gemcitabine at IC25
and IC50 concentrations for 48 h. RTKN2 expression was
quantified by real-time PCR and normalized to β-actin (ACTB).
*P<0.05, **P<0.01 and
***P<0.001. CCA, cholangiocarcinoma; RTKN2, Rhotekin
2; P, parental; GemR, gemcitabine resistant.

Figure 3

Identification of three potential
ligand-binding sites on RTKN2: Site 2 (orange), site 5 (blue), and
site 9 (green) by CavityPlus. The superimposed docked structures of
the top three screened compounds at each binding site of RTKN2 are
shown in the dashed boxes. RTKN2, Rhotekin 2.

Figure 4

2D interaction diagrams of the top
three docked candidates at each site of RTKN2. Key interactions,
including hydrogen bonds, halogen interactions, van der Waals
forces, and π interactions between each ligand and RTKN2 residues
are shown in (A) site 2, (B) site 5, and (C) site 9 of RTKN2.
RTKN2, Rhotekin 2.
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Spandidos Publications style
Kidoikhammouan S, Mahalapbutr P, Ma‑In P, Lert‑Itthiporn W, Deenonpoe R, Suriya U, Wongkham S and Seubwai W: Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma. Biomed Rep 24: 52, 2026.
APA
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U. ... Seubwai, W. (2026). Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma. Biomedical Reports, 24, 52. https://doi.org/10.3892/br.2026.2125
MLA
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U., Wongkham, S., Seubwai, W."Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma". Biomedical Reports 24.5 (2026): 52.
Chicago
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U., Wongkham, S., Seubwai, W."Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma". Biomedical Reports 24, no. 5 (2026): 52. https://doi.org/10.3892/br.2026.2125
Copy and paste a formatted citation
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Spandidos Publications style
Kidoikhammouan S, Mahalapbutr P, Ma‑In P, Lert‑Itthiporn W, Deenonpoe R, Suriya U, Wongkham S and Seubwai W: Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma. Biomed Rep 24: 52, 2026.
APA
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U. ... Seubwai, W. (2026). Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma. Biomedical Reports, 24, 52. https://doi.org/10.3892/br.2026.2125
MLA
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U., Wongkham, S., Seubwai, W."Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma". Biomedical Reports 24.5 (2026): 52.
Chicago
Kidoikhammouan, S., Mahalapbutr, P., Ma‑In, P., Lert‑Itthiporn, W., Deenonpoe, R., Suriya, U., Wongkham, S., Seubwai, W."Integrated transcriptomic and molecular docking analysis identifies Rhotekin 2 as a promising therapeutic target for overcoming gemcitabine resistance in cholangiocarcinoma". Biomedical Reports 24, no. 5 (2026): 52. https://doi.org/10.3892/br.2026.2125
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